Argonaute Proteins: Potential Biomarkers for Human Colon Cancer Lan Li1, Chaohui Yu1, Hengjun Gao2*, Youming Li1*

Li et al. BMC Cancer 2010, 10:38 http://www.biomedcentral.com/1471-2407/10/38

RESEARCH ARTICLE Open Access Argonaute proteins: potential biomarkers for human colon cancer Lan Li1, Chaohui Yu1, Hengjun Gao2*, Youming Li1*

Abstract Background: Although Argonaute proteins are considered to play important roles in stem cell self-renewal, RNA interference (RNAi) and translational regulation, relatively little is known about their functions in human disease. In this study, we investigated the expression of eight members of human Argonaute family in colon cancer and identified their potential roles in tumor development and progression. Methods: Antibodies against human Argonaute proteins were prepared by immunizing rabbits with synthetic peptides derived from the sequences of Argonaute members. Then we constructed a tissue microarray containing 75 specimens from colon cancer and 75 specimens from adjacent non-cancer tissue, and assayed eight different proteins (EIF2C1, EIF2C2, EIF2C3, EIF2C4, PIWIL1, PIWIL2, PIWIL3 and PIWIL4) by immunohistochemistry on consecutive formalin-fixed tissue microarray sections. Results: The expression of EIF2C1-4 and PIWIL1-4 was significantly higher in tumorous tissue than in adjacent tissue. Notably, a significant correlation was observed between the positive expression of EIF2C2, EIF2C3, EIF2C4, PIWIL4 and the presence of distant metastasis. Logistic regression analysis revealed that an increased expression of EIF2C1 and PIWIL2 was significantly associated with occurrence of colon cancer tissue compared with non-cancer tissue. Conclusions: Argonaute proteins are overexpressed in colon cancer relative to adjacent non-cancer tissue. The expression of EIF2C2-4 and PIWIL4 appears increased in advanced tumors with distant metastasis, suggesting it may promote tumor invasion. Furthermore, EIF2C1 and PIWIL2 might represent novel colon cancer markers with early diagnostic significance.

Background The evaluation of the clinical utility of each of these Colon cancer is one of the most frequent and lethal genes would require multiple consecutive experiments malignancies worldwide, and the 5-year survival rate is with hundreds of tumor specimens. This would be both less than 50% [1,2]. Given the high level in incidence time-consuming as well as impractical for more than a rate and mortality rate of colon cancer, it would be handful of genes. Microarray technology provides a new important to better understand the biological basis of and promising tool that allows the detection of a large tumor development and progression, to develop markers variety of parameters simultaneously, and will be of for assessing onset or prediction of therapy outcome, as importance in the fight against colon cancer. well as to identify targets for the development of novel Argonaute proteins are present in all RNA-induced therapies. Colon cancer may be considered the final step silencing complexes (RISC) reported to date and are of a progressive imbalance between mucosal cell prolif- now the best defined protein component of the RNA eration and apoptosis due to the activation of oncogenes interference (RNAi) machinery [6]. Humans have eight and the inactivation of tumor suppressor genes [3-5]. Argonaute-like proteins, four of which fall into the eIF2C/AGO subfamily (EIF2C1/hAGO1, EIF2C2/ * Correspondence: [email protected]; [email protected] hAGO2, EIF2C3/hAGO3, and EIF2C4/hAGO4) while 1Department of Gastroenterology, the First Affiliated Hospital, College of the remainders are the PIWI subfamily (PIWIL1/HIWI, Medicine, Zhejiang University, Hangzhou, 310003, China PIWIL2/HILI, PIWIL3, and PIWIL4/HIWI2) [7]. The 2Institute of Digestive Disease, Tongji Hospital affiliated to Tongji University, Shanghai, 200065, China AGO subfamily is present in animals, plants, and fission

© 2010 Li et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Li et al. BMC Cancer 2010, 10:38 Page 2 of 8 http://www.biomedcentral.com/1471-2407/10/38

yeast. Proteins of this subfamily use small interfering Table 1 Clinicopathaological characteristics of colon RNAs (siRNAs) and/or microRNAs (miRNAs) as cancer cases sequence specific guides in both transcriptional and post- Characteristic No. (n = 75) % transcriptional silencing mechanisms [8]. It is postulated Age, years that eIF2C proteins might have regulatory functions in <60 30 40 cancer stem cell self-renewal through the RNA-mediated ≥60 45 60 gene silencing mechanism as a component of RISC. In Sex contrast to the AGO subfamily, the PIWI subfamily has Female 37 49.3 been identified only in animals. The PIWI subfamily Male 38 50.7 genes are expressed mainly in germ cells, whereas AGO Duke’s stage subfamily genes are ubiquitously expressed. Consistent A912 with their expression patterns, PIWI proteins may parti- B 31 41.3 cipate in germ cell proliferation and their overexpression C 28 37.3 may cause germ cell malignancy development [9]. D 7 9.3 Although Argonaute proteins are considered to play Histologic type important roles in RNA interference, stem cell self- Adenocarcinoma 72 96 renewal and translational regulation, relatively little is Mucinous carcinoma 2 2.7 known about their functions in human disease. In the Signet-ring cell carcinoma 1 1.3 present study, we constructedatissuemicroarraycon- Histologic grade taining 150 specimens from adjacent non-cancer tissue I 8 10.7 and colon cancer tissue and assayed the expression of II 54 72 eight members of human Argonaute family by immuno- III 13 17.3 histochemistry on consecutive formalin-fixed tissue Lymph nodes metastasis microarray sections. The aim was to obtain a comprehen- Absence 44 58.7 sive survey of the expression of Argonaute proteins in Presence 31 41.3 colon cancer and identify their potential roles in tumor Distant metastasis development and progression. Absence 68 90.7 Presence 7 9.3 Methods Patients and colonic specimens We selected 75 patients with colon cancer who under- in rabbits as described by Pastor-Navarro N et al. [10]. went surgery at hospitals that cooperated with Shanghai Evolution of the antibody titer was controlled by mea- Outdo Biotech Co., Ltd. during 2005-2007. They were suring the binding of serial dilutions of the antisera to 38 men and 37 women, ranging from 25 to 85 years of microtiter plates coated with peptide antigen. Eight age (median: 57 years). Clinicopathaological characteris- weeks after the first injection, sera were collected and tics of the research subjects are shown in Table 1. Paraf- used to prepare purified antibodies. The IgG solution fin-embedded diagnostic tumor biopsy specimens and was fractionated from the rabbit antisera by precipita- their adjacent non-tumor specimens (≤ 1.5 cm away tion with 40% saturated ammonium sulphate and then from the tumor) were collected before any treatment. affinity-purified on peptide affinity columns. The flow- Patients were only included in the study if they had through was collected and stored at -20°C for the use of provided written consent to participate in the study after receiving oral and written information regarding its Table 2 Amino acid sequences of immunogens for course and purpose. Approval for the study was received preparation of antibodies against human Argonaute from the Ethics Committee of the host institution. proteins Sequences AA number Preparation of antibodies against Argonaute proteins EIF2C1 144ALVSGQIPVPLESV157-C 14 The optimal peptide immunogens for preparation of EIF2C2 137CVSLQALHDALSGR150-C 14 antibodies against human Argonaute proteins were EIF2C3 144TLPEPLELDK153-C 10 selected by an in-house peptide selection database called EIF2C4 632SQELLYSQEVIQ643-C 12 Antibody Designer, and then synthesized and fractio- PIWIL1 440DWGLSFDSNLLSFSGR455-C 16 nated by C-Strong Corporation (Shanghai, China). The PIWIL2 345DPTSAMVLQQHR356-C 12 amino acid sequences for each peptide antigen are PIWIL3 78EPGPEAGLHTAPL90-C 13 collected in Table 2. The synthetic peptide antigens PIWIL4 434WGLHFGSQISLTGR447-C 14 (KLH-coupled) were used to raise polyclonal antibodies AA: amino acid. Li et al. BMC Cancer 2010, 10:38 Page 3 of 8 http://www.biomedcentral.com/1471-2407/10/38

ELISA, Western blot, and immunohistochemistry regression analysis. A value of P < 0.05 was considered analyses. to be statistically significant.

Tissue microarray construction Results The colonic tissue microarray was constructed as Production and validation of antibodies against described previously [11]. Briefly, a tissue arraying Argonauteproteins instrument (Beecher Instruments, Silver Spring, MD) An in-house peptide selection database called Antibody was used to create holes in a recipient paraffin block Designer was used to select optimal peptide immuno- and to acquire tissue cores from the donor block by a gens for production of antibodies against human Argo- thin-walled needle with an inner diameter of 1.5 mm, naute proteins. For each protein, a synthetic peptide held in an X-Y precision guide. The cylindrical sample derived from the sequence of Argonaute member was from the selected region in the donor block was conjugated to KLH for immunization. All peptides extruded directly into the recipient block with defined showed strong immune responses after 4 immunizations array coordinates. After construction of the array in rabbits, and antisera exhibited high titers when tested block, multiple 5-μm sections were cut with a micro- by ELISA using immobilized peptides on 96-well micro- tome and placed on polylysine-coated slides. The tissue titer plates. Antibodies from individual rabbits were pur- array block contained 150 samples, including 75 ified separately by peptide affinity chromatography. tumorous specimens and 75 adjacent non-tumorous Typically 2-20 milligrams of purified antibodies were specimens. obtained for each peptide. SDS-PAGE analysis showed that the molecular mass of purified antibodies was Immunohistochemistry 50 kDa, which corresponded to the molecular mass of Immunohistochemical staining was performed using rabbit IgG. Total protein extracted from either Hela or two-step method. The sections were deparaffinized and 293 cells was used for immunoblotting with purified rehydrated. Antigen retrieval was performed by auto- antibodies. Western blot analysis showed that these claving the slides in 10 mM citric acid buffer. Antibo- purified antibodies recognized the bands at expected dies against Argonaute proteins (dilution of 1:100), and molecule mass corresponding to each Argonaute mem- goat anti-rabbit immunoglobulin/HRP from DAKO ber, respectively (Figure 1). In contrast, no band was were selected as primary antibody and secondary anti- detected with preimmune rabbit serum. Furthermore, body. Reaction products were visualized with diamino- immunohistochemical analysis showed predominantly benzidine as the chromogen and finally counterstained cytoplasmic staining in most tumorous tissues but very with haematoxylin. weak or absent staining in normal human tissues. The immunohistochemical expression of each marker was examined by light microscopy through calculating Argonaute proteins expression in adjacent non-tumorous 1000 cells per 5 sights and evaluating the average num- tissue and tumorous tissue ber. The percentage of positive cells, as the extent of By use of a large tissue microarray (150 cores) we inves- immunostaining, was quantified under microscope and tigated the protein expression of EIF2C1-4 and PIWIL1- classified into five groups. 0: < 5% positive cells; 1: 5% 4 in colon cancer specimens and adjacent non-tumorous to 24% positive cells; 2: 25% to 49% positive cells; 3: tissue. The tumorous or non-tumorous mucosa-specific 50% to 74% positive cells and 4: ≥ 75% positive cells. staining was semi-quantitatively scored by the intensity Intensity was scored as 0 for absence of staining, 1 for and the percentage of positive staining. As shown in weak, 2 for moderate, and 3 for strong staining. The Figure 2 and 3, Argonaute proteins expression was score of the intensity plus the percentage of positive detected mainly in cytoplasm of malignant cells. The staining was used to define expression levels. 0-1: nega- positive expression of EIF2C1 (P <0.001),EIF2C2(P < tive; 2-3: little positive (+1); 4-5: moderately positive 0.001), EIF2C3 (P <0.001),EIF2C4(P < 0.001), PIWIL1 (+2); 6-7: strongly positive (+3). (P <0.001),PIWIL2(P < 0.001), PIWIL3 (P < 0.001) and PIWIL4 (P < 0.001) in tumorous tissue was signifi- Statistical analysis cantly higher than in adjacent non-tumorous tissue. The differential expression of Argonaute proteins Images of representative immunostaining are presented between tumorous tissue and non-tumorous tissue was in Figure 2 and 3. The results are shown in Table 3. determined by Mann-Whitney U-test. Relationships between clinicopathological and molecular parameters Relationship between the expression of Argonaute were statistically analyzed using Spearman’s rank corre- proteins and clinicopathological parameters lation coefficient. The influence of each variable on On the basis of the above results in the colon cancer histotype of colonic lesion was assessed by logistic specimens, it would be interesting to evaluate the Li et al. BMC Cancer 2010, 10:38 Page 4 of 8 http://www.biomedcentral.com/1471-2407/10/38

Figure 1 Western blot analysis of rabbit polyclonal antibodies against Argonaute proteins in Hela or 293 cell lines.(A) Total protein extracted from Hela or 293 cells was subjected to Western blot with polyclonal antibodies against EIF2C1-4 proteins. There were the bands at expected molecule mass corresponding to each member of AGO subfamily in the films. (B) Total protein extracted from Hela or 293 cells was subjected to Western blot with polyclonal antibodies against PIWIL1-4 proteins. There were the bands at expected molecule mass corresponding to each member of PIWI subfamily in the films.

correlation between the expression of Argonaute proteins expression of EIF2C1 and PIWIL2 was significantly and clinicopathological parameters in colon cancer. As associated with occurrence of colon cancer tissue (OR = shown in Table 4, there were no statistical differences 3.071, P = 0.005 and OR = 7.392, P < 0.001, respec- among each protein expression and age, sex, histological tively) (Data are shown in Table 5). However, the rest of grade, lymph node status or Duke’s stage. The positive human Argonaute proteins had no significant effects on expression of EIF2C2 (r = 0.268, P < 0.05), EIF2C3 histotype of colonic lesion. (r = 0.269, P < 0.05), EIF2C4 (r = 0.242, P < 0.05) and PIWIL4 (r = 0.301, P < 0.01) in colon cancer was asso- Discussion ciated with the presence of distant metastasis. Here, we performed a first systematic expression analysis of human Argonaute proteins on a cohort of 75 Chi- Logistic regression analysis of influence on histotype nese colon cancer specimens and subsequently ofcolonic lesion identified potential roles for Argonaute proteins in the A stepwise forward logistic regression was used to assess development and progression of colon cancer. Because the effects of EIF2C1-4 and PIWIL1-4 on histotype of of the lack of commercially available antibodies against colonic lesion (whether the histotype of colonic lesion Argonaute proteins, we prepared eight rabbit polyclonal was tumorous tissue or adjacent non-tumorous tissue). antibodies that recognized human Argonaute proteins Logistic regression analysis revealed that an increased efficiently. With the antibodies, we detected the tissue Li et al. BMC Cancer 2010, 10:38 Page 5 of 8 http://www.biomedcentral.com/1471-2407/10/38

Figure 2 Immunohistochemical expression of EIF2C1, EIF2C2, EIF2C3 and EIF2C4 in colon cancer and adjacent non-cancer tissue.In colon cancer tissue EIF2C1-4 expression was often stronger in the cytoplasm compared with adjacent non-cancer tissue (A, B, C, D). Adjacent non-cancer tissue is detected with very weak EIF2C1-4 expression in the cytoplasm (E, F, G, H). Magnifications: ×200. distribution of EIF2C1-4 and PIWIL1-4 by immuno- the short arm of chromosome 1 in the region 1p34-p35. histochemistry on tissue microarray. This genomic region is frequently lost in human cancers Although several human Argonaute proteins have such as Wilms tumors, neuroblastoma, and carcinomas been identified, relatively little is known about their of the breast, liver, and colon [14]. The human EIF2C1 functions in human disease. AGO subfamily members gene is ubiquitously expressed at low to medium levels, are components essential for siRNA-mediated gene and EIF2C1 expression was found to be elevated in silencing in mammalian cells and involved in the effec- Wilms tumors that lacked functional copies of the ter step of mammalian RNAi [12,13]. Recent studies Wilms tumor suppressor gene WT1 [14]. Together, demonstrate that the human EIF2C1 gene is located on these findings could make human EIF2C1 an interesting

Figure 3 Immunohistochemical expression of PIWIL1, PIWIL2, PIWIL3 and PIWIL4 in colon cancer and adjacent non-cancer tissue.In colon cancer tissue PIWIL1-4 expression was often stronger in the cytoplasm compared with adjacent non-cancer tissue (A, B, C, D). Adjacent non-cancer tissue is detected with very weak PIWIL1-4 expression in the cytoplasm (E, F, G, H). Magnifications: ×200. Li et al. BMC Cancer 2010, 10:38 Page 6 of 8 http://www.biomedcentral.com/1471-2407/10/38

Table 3 EIF2C1-4 and PIWIL1-4 expression in adjacent through RNAi-related pathways or possibly also through non-tumorous tissue and tumorous tissue distinct mechanisms, AGO subfamily members have an Marker Histotypea Expression levels (number) important role in the progression of colon cancer. -+1+2+3 As a subfamily of Argonaute proteins, PIWI proteins EIF2C1 T 1 11 48 15 are expressed in the germline and in somatic cells as N3129141well [15]. Four PIWI-like proteins have been identified EIF2C2 T 1 0 30 44 in Homo sapiens. Unlike AGO subfamily proteins, N 5 32 25 13 PIWI subfamily proteins do not associate with siRNAs EIF2C3 T 1 1 49 24 and/ormiRNAs,ortheydosotoalesserextent[9]. N 10351515Recently, it has become clear that PIWI subfamily pro- EIF2C4 T 0 1 38 36 teins bind to a third class of small RNAs called PIWI- N 14391012interacting RNAs (piRNAs) [16-19]. piRNAs and PIWIs PIWIL-1 T 1 4 56 14 appear to be involved in the epigenetic control of gene N1636194expression, the control of mRNA stability, transposon PIWIL-2 T 1 1 46 27 silencing and translation regulation [6,20,21]. Elevated N2330202expression of PIWI subfamily has been reported in sev- PIWIL-3 T 3 16 47 9 eral human tumor entities. First, PIWIL1 expression has N3331101been analysed in male germline cells, showing that PIWIL1 PIWIL-4 T 1 4 49 21 mRNA levels of was upregulated in the occur- N2334117rence of seminomas, that is, a type of testicular germ cell tumors [22]. Liu et al. [23] showed that the percen- aT: Tumorous tissue; N: Non-tumorous tissue. tage of cells that expressed PIWIL1 increased from 10% candidate gene for potential involvement in neoplastic in normal gastric tissues to 76% in gastric cancer. In development. In our study, it should be noted that posi- addition, expression of the PIWIL2 protein was also tive reaction to each AGO in colon cancer tissue was found in different tumors examined, including prostate, significantly higher than that in adjacent non-cancerous breast, pancreatic, gastrointestinal, ovarian and endome- tissues. The relationship of AGO subfamily with colon trial cancer of human and in breast tumors, rhabdo- cancer has not been completely elucidated. Perhaps myosarcoma and medulloblastoma of mouse [24]. Our

Table 4 Relationship between the expression of Argonaute proteins and clinicopathological parameters Marker Correlation coefficient (r) Sex Age Duke’s stage Histologic grade LN status Distant metastasis EIF2C1 0.074 -0.082 -0.121 -0.048 -0.001 0.130 EIF2C2 0.080 0.078 0.022 0.019 -0.163 0.268a EIF2C3 0.100 -0.053 0.209 0.200 0.091 0.269a EIF2C4 0.026 0.010 0.090 0.089 -0.116 0.242a PIWIL-1 0.038 0.149 0.100 -0.135 -0.024 0.205 PIWIL-2 0.123 -0.093 -0.126 -0.025 -0.087 -0.004 PIWIL-3 -0.008 0.214 0.026 -0.003 -0.097 0.146 PIWIL-4 0.014 -0.033 0.064 -0.146 -0.111 0.301b aP <0.05,bP < 0.01, LN: lymph node.

Table 5 Logistic regression analysis of the effect of Argonaute proteins on histotype of colonic lesion Variable Expression levels No. % Tumorous tissue Wald c2 P OR 95% CI EIF2C1 - 32 3.13 +1 40 27.5 +2 62 77.42 +3 16 93.75 7.723 0.005 3.071 1.392-6.777 PIWIL2 - 24 4.17 +1 31 3.23 +2 66 69.70 +3 29 93.10 14.894 0.000 7.392 2.676-20.414 Li et al. BMC Cancer 2010, 10:38 Page 7 of 8 http://www.biomedcentral.com/1471-2407/10/38

investigation showed that the positive rate of PIWI pro- The interaction of Argonaute proteins with small tein expression in colon cancer tissue was remarkably RNAs or other part of RISC involved in carcinogenesis higher than that in non-cancer tissue. These results has not been completely elucidated. Gene specific trans- confirm the notion derived from in vitro experiments lational control induced by some miRNA species has that PIWI members might be induced by oncogenic been reported to have an effect on cancer development event [22-26]. A possible involvement of PIWI subfam- [29]. As a component of RISC, Argonaute proteins bind ily in the development and progression of colon cancer to miRNAs or piRNAs, and aberrant regulation of these is proposed. small RNAs by Argonaute proteins might induce the In order to identify markers associated with clinico- malignant phenotype of cells. SND1, also reported to be pathological characteristics of colon cancer patients, a component of RISC, is overexpressed in human colon the relationship between sex, age, histological grade, cancer tissues, even in early-stage lesions [30-32]. The metastasis, Duke’s classification, and protein expression relationship of SND1 with Argonaute proteins was still needed additional research. Previous studies rarely unclear. Paukku et al. [30] reported that the effect of considered the clinical meaning of the presence of SND1 through 3’-untranslated region of angiotensin II AGOs and PIWIs in colon cancer. In the present type 1 receptor was independent of EIF2C2, a known study, we found the positive correlation of EIF2C2, SND1 partner, and was thus RISC-independent. Identifi- EIF2C3, EIF2C4 and PIWIL4 with tumor distant cation of target mRNA species and interacting partners metastasis. It is suggested that EIF2C2-4 and PIWIL4 of Argonaute proteins might provide us with further are associated with tumor progression to advanced insights into more precise roles of Argonaute proteins stage and may promote tumor invasion. However, in colonic carcinogenesis [31]. there were no statistical differences for each protein expression among sex, age, histological grade and Conclusions Duke’sstage.Largerstudieswithahighernumberof In conclusion, this immunohistochemical study of tissue samples are necessary to confirm these results and to microarray with 75 specimens from Chinese colon can- identify the prognostic value of Argonaute proteins in cer and 75 specimens from adjacent non-tumorous tis- colon cancer. sue provided the first evidence that EIF2C1-4 and A goal of this project is to identify biomarkers occur- PIWIL1-4 were highly expressed in tumorous tissue ring in colonic carcinogenesis and contributing to colon relative to non-tumorous tissue. The expression of cancer development and progression. Of the EIF2C1-4 EIF2C2-4 and PIWIL4 appeared significantly increased and PIWIL1-4 analyzed by logistic regression, we in advanced tumors with distant metastasis, suggesting observed that an increased expression of EIF2C1 and it may promote tumor invasion. Furthermore, EIF2C1 PIWIL2 was significantly associated with occurrence of and PIWIL2 might represent novel colon cancer mar- colon cancer tissue compared with non-cancer tissue. kers with early diagnostic significance. Taking all the This is of potential clinical importance for early diagno- information together, we postulate that at least some sis. The question that remains to be discussed is how members of the human Argonaute family may be Argonaute proteins play an important role in colonic involved in the development and progression of colon carcinogenesis. It has been postulated that mutations or cancer. Further study will be required to elucidate the overexpression of several Argonaute proteins might exact biological function of each member of Argonaute cause cancer stem cells to unlimited self-renewal and family in human colon cancer. aberrant differentiation, resulting in a heterogeneous population of cells [27,28]. According to the cancer Acknowledgements stem cells hypothesis, EIF2C1 and PIWIL2 proteins The work was supported by grant from the National 863 program might play a role in the balance between colonic cancer (2002AA2Z2021). We gratefully acknowledge Xun Meng (Abmart, Shanghai) stem cells self-renewal and division in association with for support in antibody preparation. ’ small RNAs pathway. A disturbance in this balance may Author details have strong impact on neoplastic development [26]. 1Department of Gastroenterology, the First Affiliated Hospital, College of 2 Coincidentally, several previous studies have reported Medicine, Zhejiang University, Hangzhou, 310003, China. Institute of Digestive Disease, Tongji Hospital affiliated to Tongji University, Shanghai, that the gene silencing of PIWIs by RNAi or antisense 200065, China. technology inhibited the growth of cancer cells and induced cell cycle arrest in G2/M phase in human gas- Authors’ contributions LL prepared tumor tissue arrays block, carried out the immunohistochemical tric cancer and seminomas [23,24], which supported the staining, performed the statistical analysis, and drafted the manuscript. CHY issue that overexpression of Argonaute members was participated in design and preparation of antibodies, and collected the associated with proliferation and apoptosis of cancer human tissue and clinicopathological data. HJG reviewed the slides, evaluated the results of immunohistochemical staining and corrected the stem cell. Li et al. BMC Cancer 2010, 10:38 Page 8 of 8 http://www.biomedcentral.com/1471-2407/10/38

manuscript. YML designed and conducted the study. All authors read and 20. Palakodeti D, Smielewska M, Lu YC, Yeo GW, Graveley BR: The PIWI approved the final manuscript. proteins SMEDWI-2 and SMEDWI-3 are required for stem cell function and piRNA expression in planarians. RNA 2008, 14:1174-1186. Competing interests 21. Xu M, You Y, Hunsicker P, Hori T, Small C, Griswold MD, Hecht NB: Mice The authors declare that they have no competing interests. Deficient for a Small Cluster of Piwi-Interacting RNAs Implicate Piwi- Interacting RNAs in Transposon Control. Biol Reprod 2008, 79:51-57. Received: 11 April 2009 Accepted: 10 February 2010 22. Qiao D, Zeeman AM, Deng W, Looijenga LH, Lin H: Molecular Published: 10 February 2010 characterization of hiwi, a human member of the piwi gene family whose overexpression is correlated to seminomas. Oncogene 2002, References 21:3988-3999. 1. Cermak K, Thill V, Simoens CH, Smets D, Ngongang CH, da Costa PM: 23. Liu X, Sun Y, Guo J, Ma H, Li J, Dong B, Jin G, Zhang J, Wu J, Meng L, Surgical resection for colon cancer: laparoscopic assisted vs. open Shou C: Expression of hiwi gene in human gastric cancer was associated colectomy. Hepatogastroenterology 2008, 55:412-417. with proliferation of cancer cells. Int J Cancer 2006, 118:1922-1929. 2. Inoue M, Yamamoto S, Kurahashi N, Iwasaki M, Sasazuki S, Tsugane S: Japan 24. Lee JH, Schütte D, Wulf G, Füzesi L, Radzun HJ, Schweyer S, Engel W, Public Health Center-based Prospective Study Group. Daily total physical Nayernia K: Stem-cell protein Piwil2 is widely expressed in tumors and activity level and total cancer risk in men and women: results from a inhibits apoptosis through activation of Stat3/Bcl-XL pathway. Hum Mol large-scale population-based cohort study in Japan. Am J Epidemiol 2008, Genet 2006, 15:201-211. 168:391-403. 25. Grochola LF, Greither T, Taubert H, Möller P, Knippschild U, Udelnow A, 3. Grady WM, Carethers JM: Genomic and epigenetic instability in colorectal Henne-Bruns D, Würl P: The stem cell-associated Hiwi gene in human cancer pathogenesis. Gastroenterology 2008, 135:1079-1099. adenocarcinoma of the pancreas: expression and risk of tumour-related 4. Ohta M, Sugimoto T, Seto M, Mohri D, Asaoka Y, Tada M, Tanaka Y, death. Br J Cancer 2008, 99:1083-1088. Yamaji Y, Kanai F, Kawabe T, Omata M: Genetic alterations in colorectal 26. Taubert H, Greither T, Kaushal D, Würl P, Bache M, Bartel F, Kehlen A, cancers with demethylation of insulin-like growth factor II. Hum Pathol Lautenschläger C, Harris L, Kraemer K, Meye A, Kappler M, Schmidt H, 2008, 39:1301-1308. Holzhausen HJ, Hauptmann S: Expression of the stem cell self-renewal 5. Samowitz WS: Genetic and epigenetic changes in colon cancer. Exp Mol gene Hiwi and risk of tumour-related death in patients with soft-tissue Pathol 2008, 85:64-67. sarcoma. Oncogene 2007, 26:1098-1100. 6. Faehnle CR, Joshua-Tor L: Argonautes confront new small RNAs. Curr Opin 27. Yoo MH, Hatfield DL: The cancer stem cell theory: is it correct? Mol Cells Chem Biol 2007, 11:569-577. 2008, 26:514-516. 7. Sasaki T, Shiohama A, Minoshima S, Shimizu N: Identification of eight 28. Mittal S, Mifflin R, Powell DW: Cancer stem cells: the other face of Janus. members of the Argonaute family in the human genome small star, Am J Med Sci 2009, 338:107-112. filled. Genomics 2003, 82:323-330. 29. Esquela-Kerscher A, Slack FJ: Oncomirs - microRNAs with a role in cancer. 8. Carmell MA, Xuan Z, Zhang MQ, Hannon GJ: The Argonaute family: Nat Rev Cancer 2006, 6:259-269. tentacles that reach into RNAi, developmental control, stem cell 30. Paukku K, Kalkkinen N, Silvennoinen O, Kontula KK, Lehtonen JY: p100 ’ maintenance, and tumorigenesis. Genes Dev 2002, 16:2733-2742. increases AT1R expression through interaction with AT1R 3 -UTR. Nucleic 9. Kawaoka S, Minami K, Katsuma S, Mita K, Shimada T: Developmentally Acids Res 2008, 36:4474-4487. synchronized expression of two Bombyx mori Piwi subfamily genes, 31. Tsuchiya N, Ochiai M, Nakashima K, Ubagai T, Sugimura T, Nakagama H: SIWI and BmAGO3 in germ-line cells. Biochem Biophys Res Commun 2008, SND1, a component of RNA-induced silencing complex, is up-regulated 367:755-760. in human colon cancers and implicated in early stage colon 10. Pastor-Navarro N, Morais S, Maquieira A, Puchades R: Synthesis of haptens carcinogenesis. Cancer Res 2007, 67:9568-9576. and development of a sensitive immunoassay for tetracycline residues. 32. Kuruma H, Kamata Y, Takahashi H, Igarashi K, Kimura T, Miki K, Miki J, Application to honey samples. Anal Chim Acta 2007, 594:211-218. Sasaki H, Hayashi N, Egawa S: Staphylococcal nuclease domain-containing 11. Kallioniemi OP, Wagner U, Kononen J, Sauter G: Tissue microarray protein 1 as a potential tissue marker for prostate cancer. Am J Pathol technology for high-throughput molecular profiling of cancer. Human 2009, 174:2044-2050. Molecular Genetics 2001, 10:657-662. 12. Jing Q, Huang S, Guth S, Zarubin T, Motoyama A, Chen J, Di Padova F, Pre-publication history Lin SC, Gram H, Han J: Involvement of microRNA in AU-rich element- The pre-publication history for this paper can be accessed here: http://www. mediated mRNA instability. Cell 2005, 120:623-634. biomedcentral.com/1471-2407/10/38/prepub 13. Doi N, Zenno S, Ueda R, Ohki-Hamazaki H, Ui-Tei K, Saigo K: Short- doi:10.1186/1471-2407-10-38 interfering-RNA-mediated gene silencing in mammalian cells requires Cite this article as: Li et al.: Argonaute proteins: potential biomarkers Dicer and eIF2C translation initiation factors. Curr Biol 2003, 13:41-46. for human colon cancer. BMC Cancer 2010 10:38. 14. Koesters R, Adams V, Betts D, Moos R, Schmid M, Siermann A, Hassam S, Weitz S, Lichter P, Heitz PU, von Knebel Doeberitz M, Briner J: Human eukaryotic initiation factor EIF2C1 gene: cDNA sequence, genomic organization, localization to chromosomal bands 1p34-p35, and expression. Genomics 1999, 61:210-218. 15. Lin H, Spradling AC: A novel group of pumilio mutations affects the asymmetric division of germline stem cells in the Drosophila ovary. Development 1997, 124:2463-2476. 16. Girard A, Sachidanandam R, Hannon GJ, Carmell MA: A germline specific Submit your next manuscript to BioMed Central class of small RNAs binds mammalian Piwi proteins. Nature 2006, and take full advantage of: 442:199-202. 17. Grivna ST, Beyret E, Wang Z, Lin H: A novel class of small RNAs in mouse • Convenient online submission spermatogenic cells. Genes Dev 2006, 20:1709-1714. 18. Houwing S, Kamminga LM, Berezikov E, Cronembold D, Girard A, Elst van • Thorough peer review den H, Filippov DV, Blaser H, Raz E, Moens CB, Plasterk RH, Hannon GJ, • No space constraints or color ﬁgure charges Draper BW, Ketting RF: A role for Piwi and piRNAs in germ cell • Immediate publication on acceptance maintenance and transposon silencing in Zebrafish. Cell 2007, 129:69-82. 19. O’ Donnell KA, Boeke JD: Mighty Piwis defend the germline against • Inclusion in PubMed, CAS, Scopus and Google Scholar genome intruders. Cell 2007, 129:37-44. • Research which is freely available for redistribution

Submit your manuscript at www.biomedcentral.com/submit